The field of the present invention concerns compounds that react with specific sequences in proteins. The present invention more particularly concerns a class of compounds that react, under physiologic conditions, with proteins having adjacent or neighboring basic amino acid sequences. The compounds of the invention can be used to label specifically such proteins for research purposes and to disrupt their function for pharmacologic purposes. The compounds of the invention can be used for targeted inactivation of nuclear localization signal in specific proteins or molecular complexes. The compounds of the invention can also be used to treat infectious diseases such as HIV infection and malaria.
Those skilled in the art will appreciate that there are many compounds that can react with specific amino acid residues in proteins, e.g., with sulfhydryl, amino, carboxyl moieties. These reagents are substrate specific, in the sense that each reacts only with one or a few specific amino acids wherever they occur within a protein""s sequence. However, the reactivity of such reagents is not affected by the adjacent or neighboring amino acids that form the environment of the reactive moiety. Thus, the reactivity of such compounds is not context or neighborhood specific.
The function of an intracellular protein is usually the result of the overall three dimensional (tertiary) structure of the protein. However, nuclear importation is determined by the simple presence of a short sequence, called a nuclear localization signal (NLS), which functions relatively independently of its position relative to the remainder of the structure of object that is imported. In eukaryotic cells all proteins are made in the cytoplasm, which is outside of the nucleus. In general, those proteins larger than 40 kD that are specifically localized in the nucleus of the cell must be actively imported into the nucleus through the nuclear membrane from the cytoplasm via an ATP-dependent mechanism that is independent of cell division. The proteins, and other objects, that are imported have a nuclear localization signal (NLS), usually located within the NH2 terminal segment of the protein. Several such sequences are known:
a. PKKKRKV from large T antigen of SV40 and other papillomaviruses such as JC, see Kalderon, D., et al., 1984, Cell 39:499-509;
b. [AV] KRPAATKKAGQAKKKK[LD] from nucleoplasmin, in which only one of the two bracketed sequences is required, Dingwall, C., et al., 1988, J. Cell Biol. 107:841-49;
c. PRRRRSQS from hepatitis B HbcAg-Yeh, C. T., 1990, J. Virol.
d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene product p110rblxe2x80x94Zacksenhaus E. et al., 1993, Mol.Cell.Biol. 13:4588
e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1, Bukrinsky, M. I., et al., 1993, Nature 365:666.
Other viruses that contain NLS sequences include influenza virus (NP, PA, PB1, PB2 proteins which have lysine-rich NLS similar to SV40), hepatitis delta virus (HDAg, which has the sequence PKKKXKK), parvoviruses such as RA1 (NS, VP proteins which have lysine-rich NLS similar to SV40), Herpes simplex and measles virus. The recognition of an NLS sequence is largely independent of the detailed structure of the object which includes it and of its site of attachment. Goldfarb, D. S. et al., 1986, Nature 332:641-44; Lanford, R. E., 1986, Cell 46:575. Mere juxtaposition of the amino acids of the NLS is not sufficient for function, for example NLS function is generally not conferred by the peptide having the same sequence of amino acids in the opposite order as the NLS sequence. Adam, S. A. et al., 1989, Nature 337:276-79.
The primary structure, i.e., the linear sequence, of the NLS most frequently contains consecutive lysines, the Nxcex5moieties of which presumably closely approach one another, i.e., they are neighbors. However, certain functional NLS peptides lack consecutive lysines. Robbins, J., et al., 1991, Cell 64:615-23. Presumably the secondary and tertiary structure of these so called xe2x80x9cbipartitexe2x80x9d NLS peptides gives rise to neighboring NE moieties, which may be important for their activity.
Docking and subsequent movement of proteins across the nuclear pore complex require transport factors. Import of NLS-containing proteins across the nuclear pore complex is mediated by karyopherin xcex1xcex2 heterodimers (also termed NLS receptor/importin) which bind NLS-containing proteins in the cytosol and target them to the nucleus (Gorlich, D., et al., 1995, Curr. Biol. 5:383-392; Radu, A., et al., 1995, Proc. Natl. Acad. Sci. 92:1765-1773). Karyopherin xcex1 binds the NLS (Adam and Gerace, 1991, Cell 66:837-847) whereas karyopherin xcex2 enhances the affinity of xcex1 for the NLS (Rexach and Blobel, 1995, Cell 83:683-692) and mediates docking of karyopherin-NLS protein complexes to nucleoporins (a collective term for nuclear pore complex proteins) that contain FXFG peptide repeats. The GTPase Ran and its interacting protein p10 (also termed NTF2) (Moore and Blobel, 1994, Proc. Natl. Acad. Sci. 91:10212-10216) impart mobility to the translocation process by catalyzing the disruption of karyopherin xcex1xcex2 heterodimers that have docked to a nucleoporin (Nerhbass and Blobel, 1996, Science 272:120-122). Partial reactions of the nuclear import can be reproduced in vitro using solution binding assays and recombinant karyopherins (Rexach and Blobel, 1995, supra).
Two inhibitors of the nuclear localization process have been described. Nuclear localization has been inhibited by lectins (e.g., wheat germ agglutinin (WGA)) that bind to the O-linked glycoproteins associated with nuclear localization. Dabauvalle, M.-C., 1988, Exp.Cell Res. 174:291-96; Sterne-Marr R., et al., 1992, J.Cell Biol. 116:271. The nuclear localization process, which also depends upon the hydrolysis of GTP, is blocked by a non-hydrolyzable analog of GTP, e.g., (xcex3-S)GTP, Melchior, F., 1993, J.Cell Biol. 123:1649.
However, neither (xcex3-S)GTP nor WGA can be used as pharmaceuticals. Proteins, such as WGA, can be introduced into the interior of a cell only with considerable difficulty. The same limitation applies to thiotriphospates such as [xcex3-S] GTP. Further, GTPases are involved in a multitude of cell processes and intercellular signaling, thus, the use of a general inhibitor of GTPases would likely lead to unacceptable side effects.
Although HIV-1 is a retrovirus, it and other lentiviruses must be distinguished from viruses of the onco-retrovirus group, which are not associated with progressive fatal infection. For example, lentiviruses replicate in non-proliferating cells, e.g., terminally differentiated macrophages, Weinberg, J. B., 1991, J.Exp. Med. 172:1477-82, while onco-retroviruses, do not. Humphries, E. H., and Temin, H. M., 1974, J.Virol. 14:531-46. Secondly, lentiviruses are able to maintain themselves in a non-integrated, extrachromosomal form in resting T-cells. Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M. I., et al., 1991, Science 254:423; Zack, J. L., et al., 1992, J.Virol. 66:1717-25. However, it is unclear whether this phenomenon is related to the presence of latently infected peripheral blood lymphocytes (PBL) in HIV-1 infected subjects, wherein the virus is present in a provirus form. Schnittman, S. M., 1989, Science 245:305; Brinchmann, J. E., et al., 1991, J.Virol. 65:2019; Chapel, A., et al., 1992 J. Virol. 66:3966.
The productive infection of a cell by a retroviruses involves the steps of penetration into the cell, synthesis of a DNA genome from the RNA genetic material in the virion and insertion of the DNA genome into a chromosome of the host, thereby forming a provirus. Both lenti- and oncoretroviruses gain access to the host cell""s nucleus during mitosis when the nuclear membrane dissolves. However, the lentiviruses are also able to cross the nuclear membrane because viral proteins containing nuclear Focalization sequences are associated with the viral nucleoprotein complex.
The productive infection of terminally differentiated macrophages located in the central nervous system is thought to be responsible for the dementia associated with AIDS. Keonig, S., et al., 1986, Science 233:1089; Wiley, C. A. et al., 1986, Proc. Natl. Acad. Sci. 83:7089-93; Price, R. W., et al., 1988, Science 239:586-92. The infection of terminally differentiated macrophages in the lymphoid system is known to cause aberrant cytokine production. Guilian, D., et al., 1990, Science 250:1593; Fauci, A. S., et al., 1991, Ann. Int. Med. 114:678. Thus, the wasting syndrome associated with HIV-1, also known as xe2x80x9cslimxe2x80x9d disease, is believed to be a pathological process that is independent of the loss of CD4-T-cells. Rather the pathobiology of the wasting is closely related to the pathobiology of cachexia in chronic inflammatory and malignant diseases. Weiss, R. A., 1993, Science 260:1273. For these reasons, the inhibition on HIV-1 infection of macrophages and other non-dividing cells is understood to represent a highly desired modality in the treatment of HIV-1 infection, especially for patients wherein dementia or cachexia dominate the clinical picture.
Macrophages play an important role in the transmission of HIV as well. During early stages of the infection, macrophages and cells of the macrophage lineage (i.e. dendritic cells) may be the primary reservoir of HIV-1 in the body, supporting infection of T cells by antigen presentation activities, Pantaleo, G., et al., 1993, Nature 362:355-358, as well as via the release of free virus. Direct cell-to-cell transmission of the virus may constitute the major route by which infection spreads during the early stages of the disease, after resolution of the initial viremia.
It is noteworthy, in this regard, that macrophage-tropic strains of HIV-1 predominate in the early stages of infection. Thus, it appears that the infection of macrophages is particularly important during the development of a chronic infective state of the host in a newly infected subject. Secondly, macrophages are the HIV-susceptible cell type most readily passed during sexual intercourse from an HIV-infected individual into the circulation of an uninfected individual.
Finally, infection of quiescent T cells by HIV-1 has been shown to take place in vitro, Stevenson, M., et al., 1990, EMBO J. 9:1551-1560; Zack, J. A., 1990, Cell 61:213-222, and probably constitutes an important pathway for the spread of infection in vivo at various stages of the disease. Bukrinsky, M. I., et al., 1991, Science 254:423-427. Although HIV-1 does not establish productive replication in quiescent T cells, the extrachromosomal retroviral DNA can persist in the cytoplasm of such cells for a considerable period of time, and initiate replication upon activation of the host cell. Stevenson, M., et al., 1990, EMBO J. 9:1551-1560; Spina, C. A., et al., 1994, J. Exp. Med. 179:115-123; Miller, M. D., et al., 1994, J. Exp. Med. 179:101-113. A recent report suggests that the duration of viral persistence in the quiescent T cell depends on the presence of a functional NLS. von Schwedler, U., et al., 1994, Proc. Natl. Acad. Sci. 91:6992-6996. Thus, physicians recognize the desirability of preventing the infection of macrophages by HIV and understand that substantial benefits would be obtained from the use of a pharmacologic agent that prevents HIV infection in this cell type.
The mechanism whereby HIV, but not oncoretroviruses, infect non-dividing cells is now understood in broad outline. It is established that the function of the pre-integration complex of retrovirus in this regard does not depend upon the cellular mechanisms of mitosis or DNA replication, per se. Rather the integration complex must merely gain access to nucleus. Brown, P.O., et al., 1987, Cell 49:347. Oncoretroviruses gain access to the nucleus upon the dissolution of the nuclear membrane in mitosis. By contrast, lentiviruses contain two distinct proteins that mediate nuclear access through the nuclear pore complex in the absence of cellular division. For the first of these, the matrix protein (MA or p17), nuclear importation activity is clearly due to the presence of a trilysyl-containing NLS sequence. Bukrinsky, M. I., et al., 1993, Nature 365:666; von Schwedler, U., et al., 1994, Proc. Natl. Acad. Sci. 91:6992. A second protein subserving the function of nuclear entry, the vpr protein, does not contain an identifiable NLS consensus sequence. Emerman, M., et al., 1994, Nature 369:108; Heinzinger, N. K. et al., 1994, Proc. Natl. Acad. Sci. 91:7311.
The significance of the NLS sequence in the importation of HIV-1 into the nucleus of non-dividing cells has been illustrated in experiments wherein the presence in the medium of a high concentration (0.1 M) of the peptide having the sequence of the SV40 T-antigen NLS blocked the importation of HIV-1 into the nucleus of aphidicolin-arrested CD4+MT4 cells. Gulizia, J., et al., 1994, J. Virol. 68:2021-25.
Treatment of an infectious disease with chemicals involves killing or inhibition of growth of the infectious agent, which may include free-living and parasitic organisms. Parasitic diseases are widespread in the animal world where a parasitic organism lives at the expense of a host organism, and causes damage, or kills its host. Humans, domestic pets and livestocks are hosts to a variety of parasites. Parasites do not comprise a single taxonomic group, but are found within the protozoans and metazoans, among other groups. In many ways, infectious parasitic diseases resemble infectious diseases caused by microbiologicals such as fungi, bacteria and viruses.
Malaria remains one of the major health problems in the tropics. It is estimated that 300 million people a year are infected with malaria (World Health Organization, 1990, Malaria, pp. 29-40. In Tropical Diseases, Progress in Research 1989-1990, Geneva). Malaria is transmitted by Anopheles mosquitos in endemic areas, and often by blood transfusion in eradicated areas.
Malaria in humans is caused by at least four protozoan species of Plasmodium: P. falciparum, P. vivax, P. ovale and P. malariae. The asexual erythrocytic parasite, merozoite, is the stage in the life cycle that causes the pathology of malaria with a characteristic pattern of fever, chills and sweats. Anemia, acute renal failure and disturbances in consciousness are often associated with malarial infection. P. falciparum can produce a large number of parasites in blood rapidly, and causes the most morbidity and mortality.
The most important treatment of malaria to date is chemotherapy using a number of natural and synthetic drugs. Antifolates, such as pyrimethamine, inhibit the parasite""s dihydrofolate reductase, whereas the aminoquinolines, such as chloroquine (4-aminoquinoline) have the digestive vacuoles as their major site of action. Prior to the introduction of chloroquine in the 1940""s, quinine was the only effective drug for treatment of malaria. Chloroquine is commonly used to treat acute infections with all four species, but has no effect on relapses of infection by P. vivax or P. ovale. Chloroquine (500 mg weekly) may also be used to prevent malaria by suppressing the stages that multiply in the erythrocytes and cause the symptoms.
However, the use of these drugs in certain areas and in the future will be seriously hampered by the emergence of drug resistant parasites. Chloroquine resistance is widespread and will continue to appear in new areas. Due to the possibility of resistance, the presence of parasites in blood (i.e., parasitemia) is followed closely during treatment, and alternative drugs instituted if indicated. The decision on drug regimen will depend on the origin of the infection. Combination therapy, such as quinine and Fansidar (pyrimethamine and sulfadoxine), is applied to treat chloroquine-resistant P. falciparum. Because of the presence of multidrug resistant P. falciparum in many parts of the world, prevention of malaria by chemoprophylaxis with currently available drugs is not always effective.
In the last 20 years, only several drugs, such as mefloquine, halofantrine and artemisinin derivatives, have been developed to treat P. falciparum (Nosten et al., 1995, Drug Saf. 12:264-73). In view of the continuing spread of multidrug resistant P. falciparum, it is apparent that novel effective chemotherapeutic agents are needed for use against malaria.
The present invention encompasses a class of alkyl aryl carbonyl compounds that forms stable binding interactions, preferably through formation of reversible covalent bonds, with one or more basic amino acid residues, wherein such basic amino acid residues are a part of a nuclear localization signal (NLS). The stable binding interaction results in the inhibition or neutralization of the nuclear localization activity of the NLS. The binding interaction is mediated by one functional component of the compound, i.e., the reactive group, whereas another functional component of the compound, i.e. the targetting group, determines the specificity of the compound for different NLS.
This targetting function occurs by interaction of the targetting group with a docking site that is positioned proximately to the susceptible basic residues of the target NLS, such that docking of the compound places it in a favorable configuration to form a stable interaction with basic amino acid residues of the target NLS. The docking site is located either on the same NLS-bearing protein, or on another component of a larger molecular complex that includes the NLS-bearing protein.
Preferred compounds of the invention provide divalent aryl carbonyl moieties as the reactive group, particularly aryl bis(ketone), aryl bis(xcex1-diketone) or aryl bis(xcex2-diketone), linked to a targetting group, preferably to a nitrogen-containing heterocyclic functionality via an N-linkage. Particularly preferred compounds provided are bis acetyl, propanoyl, glyoxyloyl, pyruvoyl, 2-oxobutanoyl, acetoacetyl, 3-oxopentanoyl, 3-oxo-2,2-dimethylbutanoyl or 3-oxo-2,2-dimethylpentanoyl substituted aniline moieties N-linked to a pyrimidinium, pyrimidine or triazine moiety.
The invention further encompasses methods of using the compounds of the invention to form tandem binding interactions with proteins having neighboring basic residues. As used, herein, neighboring basic residues are two basic amino acid residues of a protein, particularly lysine and arginine residues, the side chain amino or guanidino functions of which approach each other as closely as the bis carbonyl functions of the arylene bis(carbonyl) compounds of the invention, when the protein is in its natured conformation. As used herein neighboring, adjacent and juxtaposed are equivalent terms in reference to amino or guanidino moieties, and refer to the physical locations of the amino or guanidino moieties in the structure of the native protein and not to the positions of the basic amino acid residues themselves in the linear sequence.
In one embodiment of the invention, the compounds of the invention can be used to react with neighboring nitrogenous moieties in lysine and arginine residues of nuclear localization sequence (NLS) of a protein, thereby inactivating the NLS. The invention also encompasses uses of compounds of the invention for specifically inhibiting importation of a NLS-containing protein or molecular complex comprising a NLS-containing protein, into the nucleus of a cell. The carbonyl group(s) of the compounds of the invention inactivates the NLS of a protein which is essential for nuclear translocation of the complex. The compounds of the invention are targeted to the complex by the specific interaction between the targetting group, preferrably a nitrogen-containing heterocyclic targetting group, and a docking site on a molecule in the complex that is distinct from the NLS. The invention further encompasses methods of screening for alkyl aryl bis(carbonyl) and bis(dicarbonyl) compounds that are capable of inhibiting importation of a specific NLS-containing protein or molecular complex comprising a NLS-containing protein into the nucleus of a cell. The screening assays of the present invention allow a compound of the present invention to be identified and selected without advance knowledge of the specific docking site that confers specificity on the NLS inhibitory activity of the compound.
The invention further encompasses methods of inhibiting productive infection by HIV-1 of terminally differentiated (non-dividing cells), particularly macrophages, by inhibition of the importation of the cytoplasmic HIV-1 complex into the nucleus of cell. Particularly the invention concerns the administration of the compound effective to block such importation to a cell. Thus, in one embodiment, the invention encompasses methods of using the above-described compounds to prevent productive infection of terminally differentiated macrophages and resting T-cells in HIV-1 infected subjects.
Without limitation as to theory, the compounds of the invention is believed to block HIV-1 replication by binding to reverse transcriptase and formation of tandem Schiff bases with neighboring NE moieties of lysines in the nuclear localization signal of HIV matrix antigen. As a result, the matrix antigen is unable to interact with karyopherin xcex1 of the host cell and the viral nucleoprotein complex does not pass across the nuclear membrane via interaction with the nuclear pore transport complex and/or other cellular components.
Moreover, compounds of the present invention are also useful for inhibiting viral infection or nuclear translocation of viral proteins in proliferating cell populations, to the extent that such occurs in some of the cells in the population during periods of the cell cycle in which the nuclear membrane is intact. Such infection or nuclear translocation of proteins in a proliferating population of cells is susceptible to treatment with the compounds of the present invention on the same basis as non-dividing or quiescent populations would be susceptible.
The invention further encompasses methods of using the compounds of the invention in treating or preventing infectious diseases such as those caused by parasites, particularly Plasmodium species that cause malaria.